Treatment of hibernating myocardium and diabetic cardiomyopathy with a GLP-1 peptide

ABSTRACT

Hibernating myocardium is characterized by viable myocardium with impaired function due to localized reduced perfusion. Hibernating myocytes retain cellular integrity, but cannot sustain high-energy requirements of contraction. High plasma levels of catecholamines, such as norepinepherine, are believed to be predictive of mortality from hibernating myocardium. Likewise, high levels of catecholamines lead to cardiomyopathy in patients with diabetes. GLP-1 reduces plasma norepinepherine levels, and it thus is useful in a method of treating hibernating myocardium or diabetic cardiomyopathy.

[0001] This application claims priority to U.S. Application Ser. Nos.60/241,834, filed Oct. 20, 2000, 60/60/242,139, filed Oct. 23, 2000, and60/245,234, filed Nov. 3, 2000, all of which are hereby incorporated byreference.

BACKGROUND OF THE INVENTION

[0002] Heart failure continues to be a major health problem.Approximately four million persons in the U.S. population have heartfailure. With a steadily aging population, four hundred thousandindividuals experience new onset heart failure each year, with a fiveyear mortality rate approaching fifty percent.

[0003] Rather than a single pathological entity, “heart failure” definesa clinical syndrome with many different etiologies that reflects afundamental abnormality in effective mechanical performance of theheart, such that the heart is unable to meet the demands of the body.There are various forms of heart failure, including “forward” and“backward” heart failure. Backward failure, synonymous with congestiveheart failure, is due to increase in venous pressure (i.e., increase inpressure in the veins that return blood to the heart) resulting from theinability of the heart to discharge its contents normally, leading topulmonary and systemic congestion. By contrast, forward failure iscaused by an inability of the heart to maintain normal tissue perfusion,resulting in fatigue, weakness, loss of weight, and impairment ofcerebral function.

[0004] Hibernating Myocardium

[0005] “Hibernating myocardium” constitutes a significant fraction offorward heart failures, and it may or may not be accompanied bypulmonary or systemic congestion. This condition reflects localizeddepressed myocardial function as a result of chronic non-criticalischemia (hypoxia resulting from low blood supply). The degree ofischemia is not sufficient to produce necrosis (infarction), but itlocally restricts myocardial oxygenation and fuel supply, such that apart of the myocardium becomes hypoactive or dormant. Hibernatingmyocytes remain viable but do not contribute to the pumping action ofthe heart. The severity of myocardial damage depends on the duration ofhibernation. Eventually, the damage may become irreversible and may leadto heart failure when the extent of myocardial dysfunction is greatenough to compromise cardiac performance and reduce the cardiac output;that is, ischemic cardiomyopathy may be the ultimate result ofhibernating myocardium, if it is not treated appropriately.

[0006] Traditionally, hibernating myocardium has been treated bysurgical revascularization through coronary bypass surgery orangioplasty. The inconvenience of surgery and the incidence of morbidityor restenosis associated with these techniques underscores the need forsupplemental or alternative pharmacological intervention. Fath-Ordoubadiet al., Heart 82: 210-216 (1999) and Pagano et al., Curr. Opin. Cardiol.14: 506-509 (1999). Effective pharmacological intervention would beespecially useful where surgery is contraindicated, as in the case ofmild hibernating myocardium, or where the patient's condition isconsidered too serious for surgery.

[0007] Congestive heart failure was first treated pharmacologically withvasodilators and inotropic agents, which increase cardiac musclecontractility. See WO 99/40788. While these drugs improved hemodynamicsover the short term, recent studies have found a discrepancy betweenimproved hemodynamics and clinical outcome. In fact, the only riskfactor found predictive of morbidity associated with congestive heartfailure is the plasma level of the catecholamine norepinepherine. Cohnet al., “Plasma norepinepherine as a guide to prognosis in patients withchronic congestive heart failure.” N. Engl. J. Med. 311: 819-823 (1984);Lahiri et al., J. Cardio. Pharm. 33 (Suppl. 3): S9-S16 (1999). Thus, inthe case of congestive heart failure, long term administration ofinotropic agents is contraindicated. The compounds most useful to treatcongestive heart failure have proven to be ACE inhibitors, which have avasodilating effect, and multi-functional β-blockers like carvedilol,which exert an anti-adrenergic effect. Lahiri et al. (1999).

[0008] Like congestive heart failure, there is evidence thatadministration of inotropic agents may worsen ischemia associated withhibernating myocardium. In one study, low level treatment with theinotrope dobutamine increased myocardial function in hibernatingmyocardium, but high levels of dobutamine increased myocardial demand tothe point where it passed an ischemic threshold. Senioer et al.,“Enhanced detection of myocardial ischaemia by stress dobutamineechocardiography utilising the ‘biphasis’ response of wall thickeningduring low and high dose dobutamine infusion.” J. Am. Coll. Cardiol. 26:26-32 (1995). This and similar studies have raised questions about thelong term benefit to mortality from inotropic agents, despite theirshort term hemodynamic benefit. In particular, it has been proposed thatfurther increases in myocardial demand may enhance ishemia associatedwith hibernating myocardium, thereby exacerbating necrosis andapoptosis. Lahiri et al. (1999).

[0009] Accordingly, it has been suggested that inotropic agents also arecontraindicated for hibernating myocardium, and that hibernatingmyocardium should be treated with the same non-inotropic, oranti-adrenergic, agents that are used to treat congestive heart failure.By analogy to congestive heart failure, it has also been suggested thathigh plasma levels of catecholamines, like norepinepherine, aredeleterious to clinical outcome of hibernating myocardium, because oftheir inotropic properties. Lahiri et al. (1999).

[0010] As there are only a handful of agents known to have limitedefficacy for the long term treatment of hibernating myocardium, thereremains a strong need for new therapeutic agents which have thepotential to revitalize hibernating cells. In particular, there remainsa strong need to find agents that can reduce the plasma blood level ofcatecholamines.

[0011] Diabetic Cardiomyopathy

[0012] Patients with diabetes are at high risk for developing diabeticcardiomyopathy (DCM). The exact etiology of this disease remainscontroversial, in part because many myocardial abnormalities areassociated with diabetes. DCM is clearly defined, however, as areversible cardiomyopathy that occurs in the absence of coronaryatherosclerosis. Bell, Diabetes Care 18: 708-714 (1995). DCM is furthercharacterized by myocardial fibrosis, that can be partially attributableto ischemia. Id. Hypertension, also characteristic of diabetes, canaggravate fibrosis to the point where DCM can become a serious, evenfatal, condition. Id.

[0013] This hypertension is at least in part due to an abnormalactivation of the sympathetic nervous system. Pallab et al., Am. J.Physiol. 252: E734-739. Among the manifestations of this aberrantactivation is an increase in the level of norepinepherine in the heart,as well as its altered metabolism by the heart. Id. High levels ofcatecholamines, such as norepinepherine, in the heart or circulationresult in the development of DCM. The accompanying myocardial damage isbelieved to be in part caused by the oxidative breakdown products ofnorepinepherine. Id. An ideal anti-hypertension agent for the diabeticpatient thus would reduce the activation of the sympathetic nervoussystem without worsening hyperglycemia or hypoglycemia. Presently, veryfew compounds provide these characteristics.

SUMMARY OF THE INVENTION

[0014] Administration of GLP-1 has been found unexpectedly to suppressplasma blood levels of norepinepherine. By analogy to congestive heartfailure, reduction in plasma norepinepherine levels will be expected toease the ischemic stress to hibernating myocardium, thereby improvingthe clinical outcome. Accordingly, administration of GLP-1 will beuseful in a method to treat hibernating myocardium, either alone or inconjunction with existing treatment regimes. Likewise, GLP-1 will beuseful in reducing norepinepherine levels in the heart and/or plasmathat are associated with the development of diabetic cardiomyopathy.

[0015] GLP-1 reduces plasma norepinepherine levels in a method oftreating hibernating myocardium or diabetic cardiomyopathy. Thus, amethod for treating hibernating myocardium or diabetic cardiomyopathycomprises administering a therapeutically effective amount of a GLP-1molecule to said patient. A GLP-1 molecule also may be administered intherapeutically effective amount to a patient suffering from congestiveheart failure or ischemic cardiomyopathy, particularly one who also hashibernating myocardium. A therapeutically effective amount of a GLP-1molecule reduces the plasma and/or heart norepinepherine level. A GLP-1molecule preferably is delivered intravenously or subcutaneously. Theformer is preferred for acute treatment with a GLP-1 molecule, while thelatter is preferred in chronic treatment regimens.

[0016] Preferred GLP-1 molecules of the invention include GLP-1(7-36)amide, GLP-1(7-37), and exendin-4. GLP-1 molecules include thosemolecules that specifically bind to and activate the GLP-1 receptor.

BRIEF DESCRIPTION OF THE FIGURES

[0017] FIGS. 1-4 summarize the results obtained from two representativeanimals, Dog A (treatment) and Dog B (placebo).

[0018]FIG. 1 demonstrates changes in left ventricular (LV)contractility, as measured by the rate of change of LV pressure (dP/dt).

[0019]FIG. 2 demonstrates changes in LV ejection fraction (EF), asmeasured by percent emptying of the LV during systole.

[0020]FIG. 3 illustrates LV contraction, as reflected by the degree ofwall thickening.

[0021]FIG. 4 reflects changes in overall cardiac function, as measuredby cardiac output (CO), which is the volume of blood (in mL) pumped perminute.

DETAILED DESCRIPTION

[0022] The present invention provides novel methods and compositions fortreatment of hibernating myocardium (HM). In particular, the presentinvention includes a method of treating a patient with HM byadministering a therapeutically effective amount of a GLP-1 molecule tothe patient. The present inventors have surprisingly discovered that inmammals suffering from HM, administration of a GLP-1 molecule resultedin rapid recovery of heart function, compared with non-treated subjects.This recovery is associated with an unexpected decrease in the plasmalevels of norepinepherine in treated mammals.

[0023] As used in this application “congestive heart failure” (“CHF”)denotes a condition characterized by an increase in venous pressure thatresults from the inability of the heart to discharge its contentsnormally, leading to pulmonary and systemic congestion. The heart muscleof a patient with CHF has a reduced ability to act as a pump. CHF isaccompanied by circulatory and neurohumoral changes which result infailure to deliver sufficient blood and oxygen supply to peripheraltissues and vital organs.

[0024] “Hibernating myocardium” means viable myocardium with impairedfunction due to reduced perfusion. HM retains cellular integrity, butcannot sustain high-energy requirements of contraction. HM isdistinguished from infarcted myocardium, which is irreversiblemyocardial damage with formation of a scar, and from stunned myocardium,which is myocardium with contractile dysfunction despite normalizationof perfusion. Jadvar et al., RadioGraphics 19: 915-926 (1999).

[0025] Clinically, HM may be detected by the use of dobutamine stressechocardiography. Shan et al., In Cardiology Clinics, G. Aurigemma, ed.,W.B. Saunders Co., Philadelphia, Vol. 17, No. 3, pages 539-553 (1999).HM may also be detected by cardiac positron emission tomography (PET),which is more accurate than single photon emission tomography (SPECT).PET with 2-(fluorine-18) fluoro-2-deoxy-D-glucose is considered thestandard of reference for determining regional or left ventricularfunction, following revascularization, to identify viable hibernatingmyocardium. Stress magnetic resonance imaging has been used to furtherdiagnose hibernating myocardium and distinguish this disease from othermyocardial disease states. HM is characterized by decrease in leftventricular (LV) function that is moderate, compared to the severedecrease associated with irreversible dysfunction or scarring. Thedegree of systolic wall thickening (SWT) is also characteristic ofmyocardial hibernation. SWT is severely decreased at rest, compared tonormal or irreversibly damaged or scarred myocardium, and SWTdysfunction distinctively improves during stress. Sensky et al.,Radiology 215: 608-614.

[0026] “Diabetic cardiomyopathy” (DCM) is defined as a reversiblecardiomyopathy in diabetics that occurs in the absence of coronaryatherosclerosis. Bell, Diabetes Care 18: 708-714 (1995); Fein, DiabetesCare 13: 1169-1179 (1990). DCM is characterized by myocardialhypertrophy and fibrosis. Microvascular pathology is also present, and,in some cases, both congestive and restrictive cardiomyopathies arepresent. Id.

[0027] The “paced dog” model, used in the Example below, provides asystem to study HM, because the exertion of the heart exceeds theheart's ability to respond, which creates an energy-limited situation.Other suitable animal models are available to study chronicallydysfunctional viable myocardium, in dogs and pigs, for example, whichallow laboratory study of therapeutic regimens. For example, the fixedLAD (left anterior descending artery) stenosis model in pigsdemonstrates cardiac dysfunction with reduced myocardial perfusion thatis analogous to humans with HM in the absence of infarction. Canty etal., Am. J. Physiol. 277: H417-H422 (1999). Similar animal models indiabetic dogs, mice and rats are available for the study of DCM. Bell(1995); Fein (1990).

[0028] A “GLP-1 molecule” includes the following. Mammalian GLP peptidesand glucagon are encoded by the same gene. In the ileum, the phenotypeis processed into two major classes of GLP peptide hormones, namelyGLP-1 and GLP-2. GLP-1 (1-37) has the sequence His Asp Glu Phe Glu ArgHis Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln AlaAla Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly (SEQ ID NO:1). GLP-1(1-37) is amidated by post-translational processing to yield GLP-1(1-36) NH₂ which has the sequence His Asp Glu Phe Glu Arg His Ala GluGly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala Lys GluPhe Ile Ala Trp Leu Val Lys Gly Arg (NH₂) (SEQ ID NO:2); or isenzymatically processed to yield GLP-1 (7-37) which has the sequence HisAla Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala AlaLys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly (SEQ ID NO:3). GLP-1(7-37) can also be amidated to yield GLP-1 (7-36) amide which is thenatural form of the GLP-1 molecule, and which has the sequence His AlaGlu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala LysGlu Phe Ile Ala Trp Leu Val Lys Gly Arg (NH₂) (SEQ ID NO:4) and in thenatural form of the GLP-1 molecule. Likewise, GLP-1(1-36) (NH₂) can beprocessed to GLP-1 (7-36) (NH₂).

[0029] Intestinal L cells secrete GLP-1 (7-37) (SEQ ID NO:3) andGLP-1(7-36)NH₂ (SEQ ID NO: 4) in a ratio of 1 to 5, respectively. Thesetruncated forms of GLP-1 have short in situ half-lives, i.e., less than10 minutes, and are inactivated by an aminodipeptidase IV to yield GluGly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala Lys GluPhe Ile Ala Trp Leu Val Lys Gly Arg Gly (SEQ ID NO:5); and Glu Gly ThrPhe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala Lys Glu Phe IleAla Trp Leu Val Lys Gly Arg (NH₂) (SEQ ID NO:6); respectively. Thepeptides Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln AlaAla Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly (SEQ ID NO:5) andGlu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala LysGlu Phe Ile Ala Trp Leu Val Lys Gly Arg (NH₂) (SEQ ID NO:6), have beenspeculated to affect hepatic glucose production, but do not stimulateproduction or release of insulin from the pancreas.

[0030] As used in this specification, the term “GLP-1 molecule” includesGLP-1 (1-37), GLP-1 (1-36) NH₂, GLP-1 (7-37), GLP-1 (7-36) NH₂ (“GLP-1(7-36) amide”) (collectively referred to as “GLP-1 peptides”). Thepresent invention includes the use of recombinant human GLP-1 peptidesas well as GLP-1 peptides derived from other species, whetherrecombinant or synthetic.

[0031] “GLP-1 molecule” further denotes biologically active variants,analogs and derivatives of GLP-1 peptides. “Biologically active,” inthis context, means having GLP-1 (7-36) biological activity, but it isunderstood that the activity of the variant can be either less potent ormore potent than native GLP-1 (7-36) amide. GLP-1 (7-36) amide is anative, biologically active form of GLP-1. See Göke et al., DiabeticMedicine. 13:854-860 (1996). GLP-1 molecules of the present inventioninclude polynucleotides that express agonists of GLP-1, i.e. activatorsof the GLP-1 receptor molecule and its secondary messenger activityfound on insulin-producing β-cells, among others. GLP-1 mimetics thatalso are agonists of GLP-1 receptors on β-cells include, for example,chemical compounds specifically designed to activate the GLP-1 receptor.

[0032] GLP-1 molecule biological activity can be determined by in vitroand in vivo animal models and human studies as is well known to theskilled artisan. Included as GLP-1 molecules are any molecules, whetherthey be peptides, peptide mimetics, or other molecules that bind to oractivate a GLP-1 receptor, such as the GLP-1 (7-36) amide receptor, andits second messenger cascade. GLP-1 receptors are cell-surface proteinsfound, for example, on insulin-producing pancreatic β-cells. The GLP-1(7-36) receptor has been characterised in the art. Methods ofdetermining whether a chemical or peptide binds to or activates a GLP-1receptor are known to the skilled artisan and are preferably carried outwith the aid of combinatorial chemical libraries and high throughputscreening techniques. GLP-1 molecules include species havinginsulinotropic activity and that are agonists of the GLP-1 receptormolecule and its second messenger activity on insulin producing β-cells,among others.

[0033] GLP-1 biological activity can be determined by standard methods,in general, by receptor-binding activity screening procedures whichinvolve providing appropriate cells that express the GLP-1 receptor ontheir surface, for example, insulinoma cell lines such as RINmSF cellsor INS-1 cells. See Mosjov, Int. J. Peptide Protein Res. 40: 333-343(1992) and EP 708170. Cells that are engineered to express a GLP-1receptor also can be used. In addition to measuring specific binding oftracer to membrane using radioimmunoassay methods, cAMP activity orglucose dependent insulin production can also be measured. In onemethod, a polynucleotide encoding the receptor of the present inventionis employed to transfect cells to thereby express the GLP-1 receptorprotein. Thus, for example, these methods may be employed for screeningfor a receptor agonist by contacting such cells with compounds to bescreened and determining whether such compounds activate the receptorand generate a signal.

[0034] Polyclonal and monoclonal antibodies can be utilized to detectpurify and identify GLP-1 like peptides for use in the methods describedherein. Antibodies such as ABGA1178 detect intact unspliced GLP-1 (1-37)or N-terminally-truncated GLP-1 (7-37) or (7-36) amide. Other antibodiesdetect on the very end of the C-terminus of the precursor molecule, aprocedure which allows by subtraction to calculate the amount ofbiologically active truncated peptide, such as GLP-1 (7-37) amide. SeeOrskov et al., Diabetes 42: 658-661 (1993) and Orskov et al., J. Clin.Invest. 87: 415-423 (1991).

[0035] Other screening techniques include the use of cells which expressthe GLP-1 receptor, for example, transfected CHO cells, in a systemwhich measures extracellular pH or ionic changes caused by receptoractivation. For example, potential agonists may be contacted with a cellwhich expresses the GLP-1 protein receptor and a second messengerresponse, e.g. signal transduction or ionic or pH changes, may bemeasured to determine whether the potential agonist is effective.

[0036] Agonists of glucagon-like peptide that exhibit activity throughthe GLP-1 (7-36) amide receptor have been described in EP 0708179;Hjorth et al., J. Biol. Chem. 269 (48): 30121-30124 (1994); Siegel etal., Amer. Diabetes Assoc. 57^(th) Scientific Sessions, Boston (1997);Hareter et al., Amer. Diabetes Assoc. 57^(th) Scientific Sessions,Boston (1997); Adelhorst et al., J. Biol. Chem. 269(9): 6275-6278(1994); Deacon et al., 16^(th) International Diabetes FederationCongress Abstracts, Diabetologia Supplement (1997); Irwin et al., Proc.Natl. Acad. Sci. USA. 94: 7915-7920 (1997); Mosjov, Int. J. PeptideProtein Res. 40: 333-343 (1992). See also Göke et al., Diabetic Medicine13: 854-860 (1996). Recent publications disclose Black Widow GLP-1 andSer² GLP-1. See Holz et al., Comparative Biochemistry and Physiology,Part B 121: 177-184 (1998) and Ritzel et al., “A synthetic glucagon-likepeptide-1 analog with improved plasma stability,” J. Endocrinol. 159(1):93-102 (1998).

[0037] “GLP-1 molecules” also include peptides that are encoded bypolynucleotides that express biologically active GLP-1 variants asdefined herein. Also included in the present invention are GLP-1molecules that are peptides containing one or more amino acidsubstitutions, additions or deletions, compared with GLP-1 (7-36) amide.In one embodiment, the number of substitutions, deletions, or additionsis 30 amino acids or less, 25 amino acids or less, 20 amino acids orless, 15 amino acids or less, 10 amino acids or less, 5 amino acids orless or any integer in between these amounts. In one aspect of theinvention, the substitutions include one or more conservativesubstitutions. A “conservative” substitution denotes the replacement ofan amino acid residue by another, biologically active similar residue.Examples of conservative substitution include the substitution of onehydrophobic residue, such as isoleucine, valine, leucine or methioninefor another, or the substitution of one polar residue for another, suchas the substitution of arginine for lysine, glutamic for aspartic acids,or glutamine for asparagine, and the like. The following table listsillustrative, but non-limiting, conservative amino acid substitutions.!ORIGINAL RESIDUE? EXEMPLARY SUBSTITUTIONS ALA SER, THR ARG LYS ASN HIS,SER ASP GLU, ASN CYS SER GLN ASN, HIS GLU ASP, GLU GLY ALA, SER HIS ASN,GLN ILE LEU, VAL, THR LEU ILE, VAL LYS ARG, GLN, GLU, THR MET LEU, ILE,VAL PHE LEU, TYR SER THR, ALA, ASN THR SER, ALA TRP ARG, SER TYR PHE VALILE, LEU, ALA PRO ALA

[0038] It is further understood that GLP-1 peptide variants include theabove described peptides which have been chemically derivatized oraltered, for example, peptides with non-natural amino acid residues(e.g., taurine residue, beta and gamma amino acid residues and D-aminoacid residues), C-terminal functional group modifications such asamides, esters, and C-terminal ketone modifications and N-terminalfunctional group modifications such as acylated amines, Schiff bases, orcyclization, such as found for example in the amino acid pyroglutamicacid.

[0039] Also included in the present invention are peptide sequenceshaving greater than 50 percent sequence identity, and preferably greaterthan 90 percent sequence identity to (1) SEQ ID NOS:1, 2, 3, 4; and (2)to truncated sequences thereof. As used herein, sequence identity refersto a comparison made between two molecules using standard algorithmswell known in the art. The preferred algorithm for calculating sequenceidentity for the present invention is the Smith-Waterman algorithm,where SEQ ID NO:1 is used as the reference sequence to define thepercentage identity of polynucleotide homologs over its length. Thechoice of parameter values for matches, mismatches, and inserts ordeletions is arbitrary, although some parameter values have been foundto yield more biologically realistic results than others. One preferredset of parameter values for the Smith-Waterman algorithm is set forth inthe “maximum similarity segments” approach, which uses values of 1 for amatched residue and −⅓ for a mismatched residue (a residue being eithera single nucleotide or single amino acid) (Waterman, Bulletin ofMathematical Biology 46:473-500 (1984)). Insertions and deletions(indels), x, are weighted as

x _(k)=1+k/3,

[0040] where k is the number of residues in a given insert or deletion(Id.).

[0041] For instance, a sequence that is identical to the 42 amino acidresidue sequence of SEQ ID NO:1, except for 18 amino acid substitutionsand an insertion of 3 amino acids, would have a percent identity givenby:

[(1×42 matches)−(⅓×18 mismatches) −(1+3/3 indels)]/42=81% identity.

[0042] Also included in “GLP-1 molecules” of the present invention aresix peptides in Gila monster venoms that are homologous to GLP-1. Theirsequences are compared to the sequence of GLP-1 in Table 1. TABLE 1Position 1 a. H A E G T F T S D V S S Y L E G Q A A K E F I A W L V K GR (NH₂) b. H S D G T F T S D L S K Q M E E E A V R L F I E W L K N G G PS S G A P P P S (NH₂) c.                 D L S K Q M E E E A V R L F I EW L K N G G P S S G A P P P S (NH₂) d. H G E G T F T S D L S K Q M E E EA V R L F I E W L K N G G P S S G A P P P S (NH₂) e. H S D A T F T A E YS K L L A K L A L Q K Y L E S I L G S S T S P R P P S S f. H S D A T F TA E Y S K L L A K L A L Q K Y L E S I L G S S T S P R P P S g. H S D A IF T E E Y S K L L A K L A L Q K Y L A S I L G S R T S P P P (NH₂) h. H SD A I F T Q Q Y S K L L A K L A L Q K Y L A S I L G S R T S P P P (NH₂)a = GLP-1 (7-36) amide (SEQ. ID NO: 4) b = Exendin 3 (SEQ. ID NO: 7). c= Exendin 4 (9-39 (NH₂(SEQ. ID NO: 8). d = Exendin 4 (SEQ. ID NO: 9). e= Helospectin I (SEQ. ID NO: 10). f = Helospectin II (SEQ. ID NO: 11). g= Helodermin (SEQ. ID NO: 12). h = Q⁸, Q⁹ Helodermin (SEQ. ID No: 13).

[0043] Peptides (a, b, d, e, f and g) are homologous in positions 1, 7,11 and 18. GLP-1 and exendins are further homologous in positions, 4, 5,6, 8, 9, 15, 22, 23, 25, 26 and 29. In position 2, A, S and G arestructurally similar. In position 3, residues D arid E (Asp and Glu) arestructurally similar. In positions 22 and 23, F (Phe) and I (Ile) arestructurally similar to Y (Tyr) and L (Leu), respectively. Likewise, inposition 26, L and I are structurally equivalent.

[0044] Thus, of the 30 residues of GLP-1, exendins 3 and 4 are identicalin 15 positions and equivalent in 5 additional positions. The onlypositions where radical structural changes are evident are at residues16, 17, 19, 21, 24, 27, 28 and 30. Exendins also have 9 extra residuesat the carboxyl terminus.

[0045] The GLP-1 molecules of the invention that are peptides that canbe made by solid state chemical peptide synthesis. Such peptides canalso be made by conventional recombinant techniques using standardprocedures described in, for example, Sambrook et al., “MolecularCloning, a Laboratory Manual,” Cold Spring Harbor Press, N.Y (1989).“Recombinant”, as used herein, means that a gene is derived from arecombinant (e.g., microbial or mammalian) expression system which hasbeen genetically modified to contain polynucleotide encoding a GLP-1molecule as described herein.

[0046] The GLP-1 like peptides can be recovered and purified fromrecombinant cell cultures by methods including, but not limited to,ammonium sulfate or ethanol precipitation, acid extraction, anion orcation exchange chromatography, phosphocellulose chromatography,hydrophobic interaction chromatography, affinity chromatography,hydroxylapatite chromatography and lectin chromatography. Highperformance liquid chromatography (HPLC) can be employed for finalpurification steps.

[0047] The GLP-1 molecule peptides of the present invention may be anaturally purified product, or a product of chemical syntheticprocedures, or produced by recombinant techniques from prokaryotic oreukaryotic hosts (for example, by bacteria, yeast, higher plant, insectand mammalian cells in culture or in vivo). Depending on the hostemployed in a recombinant production procedure, the polypeptides of thepresent invention are generally non-glycosylated, but may beglycosylated. Particularly preferred GLP-1 molecules of the inventionare GLP-1(7-36) amide, and GLP-1(7-37) and exendin-4.

[0048] Therapeutic Methods

[0049] The therapeutic methods of the invention are useful for treatingany patient suffering from HM. Such a patient also may suffer fromcongestive heart failure. Alternately, such a patient may suffer from,or be predisposed to, DCM. Typically, a GLP-1 molecule of the inventionwill be administered in a parenteral formulation. Other well knownmethods for administering a GLP-1 molecule to a patient suffering fromHM also can be employed in the methods of the invention. Theseadministration methods include, but are not limited to, subcutaneous ormicropressure injection, external or implant pump, depot injection, andother types of prolonged application dispensing devices. Other methodsof administration, such as transdermal or transmembrane administration,using patch or buccal means, also can be employed. Oral administrationalso may be suitable. Pulmonary administration, such as inhalation, alsocan be employed.

[0050] The route of administration may be optimized for particulartreatments regimens. If chronic treatment of HM is required, forexample, administration preferably will be via continuous subcutaneousinfusion, using an external infusion pump. By contrast, if acutetreatment of HM is required, as in the case of associated heart failure,then intravenous infusion is preferred.

[0051] The timing of administration of a GLP-1 molecule will depend onthe nature of the condition being treated. Administration of a GLP-1molecule may be as soon as HM or DCM is diagnosed, and theadministration can be either continuous or on an intermittent basis, foras long as necessary. For acute conditions, where heart failure suddenlyworsens, several hours to several days of continuous infusion arepreferred. For chronic treatment, a GLP-1 molecule may be administeredfor weeks to months, even years, by continuous infusion.

[0052] The amount of a GLP-1 molecule that should be administered willvary according to the severity of the conditions and the patient. Anadvantage of using GLP-1 (7-36) amide is that high doses can be usedwithout consequent hypoglycemia, because the action of GLP-1 (7-36)amide is dependent on glucose levels. Therefore, doses of up to 10.0nmol/kg can be used without adverse effects. For intravenousadministration, a typical dose of a GLP-1 molecule will be 1.5pmol/kg/min. The range of the dose may vary between about 0.1-10pmol/kg/min. For subcutaneous administration, the optimal dose is 5pmol/kg/min, with a range between about 0.5-50 pmol/kg/min.

[0053] GLP-1 can also be co-administered with other therapeutic agentsthat are known for treating HM or DCM. For HM, these therapeutic agentsinclude carvedilol, ACE inhibitors, and other anti-HM drugs, such asnitrates and hydralazine, bisoprolol, and metoprolol. See Lahiri et al.GLP-1 can be administered as an adjunct to surgerical treatment of HM,by cardiac by-pass surgery or by angioplasty, for example.Administration of GLP-1 may be made to an individual before, during orfollowing surgical treatment. Where surgery is not indicated or isundesirable, GLP-1 may be administered as an alternative treatmentregime. Treatment with GLP-1 would be especially useful, not only whensurgery is contraindicated, as in the case of mild hibernatingmyocardium, but also when the patient's condition is considered tooserious for surgery. ACE inhibitors likewise are among the preferredcompounds for treating DCM. Bell (1995).

[0054] “Treating” embraces the amelioration of an existing condition.The skilled artisan would understand that treatment does not necessarilyresult in the complete absence or removal of symptoms. Treatment alsoembraces palliative effects: that is, those that reduce the likelihoodof a subsequent medical condition. The alleviation of a condition thatresults in a more serious condition is encompassed by this term. Amethod to treat diabetic cardiomyopathy thus may comprise a method toreduce plasma norepinepherine levels in a diabetic patient, since thelatter may lead to or aggravate cardiomyopathy.

EXAMPLE 1

[0055] Beagle dogs were fitted with telemetry devices that permitlong-term ambulatory data collection in conscious animals. These devicesmeasured LV pressure, myocardial oxygen consumption (MVO₂, an expressionof myocardial efficiency), coronary flow (CBF), and cardiac output (CO).The dogs were “paced,” such that heart rate was forced up to about 240beats per minute, for 3-4 weeks, which induces moderate HM in apredictable manner. This HM dog model is an accepted model for assessingthe effectiveness of treatments for HM. Kiuchi et al., “Myocardialbeta-adrenergic receptor function during the development ofpacing-induced heart failure.” J. Clin. Invest. 91: 907-914 (1993).

[0056] Following induction of HF, five dogs were given an intravenousinfusion of rGLP-1 (7-36) amide (1.5 pmol/kg/min) for 48 hours and fourdogs served as controls. During the treatment period, “pacing” wasdiscontinued. Plasma catecholamines were assessed before and afterinfusion, along with LV pressures, coronary and systemic hemodynamics,and MVO₂. The results are summarized in Table 1. GLP-1 treatmentsignificantly reduced (*p<0.05) plasma norepinepherine (NE) levels from2.30±0.15 nmol/ml to 1.62±0.11 nmol/ml. Moreover, GLP-1 treatmentsignificantly (*p<0.05) increased left ventricular pressure (LVP), leftventricular contractility (LV dP/dt), cardiac output (CO), coronaryblood flow (CBF), and myocardial oxygen consumption (MVO₂), whilesignificantly decreasing LV end-diastolic pressure (LVEDP). These dataindicate that the rGLP-1-treated dogs demonstrated a remarkable recoveryof heart function within 48 hours of GLP-1 treatment. This wasassociated with increases in oxidative phosphorylation as measured byMVO₂, suggesting improved myocardial energetics. Thus, GLP-1 infusion isassociated with decreased plasma NE and significant improvement inmyocardial energetics. The placebo-treated control dogs did not, in thisstudy, show the same degree of heart failure as the GLP-1 group beforetreatment. However, the control animals clearly had compromisedhemodynamics, which did not improve during the 48-hour placebo treatmentperiod.

[0057] FIGS. 1-4 summarize the results obtained from two representativeanimals, Dog A (treatment) and Dog B (placebo). FIG. 1 reflects changesin left ventricular (LV) contractility, as measured by the rate ofchange of LV pressure (dP/dt). In the treated animal (dog A), pacingreduced contractility by 60%, as expected in a model of HM. Remarkably,24 hours of GLP-1 treatment restored contractility to 80% of baseline,and 48 hours of treatment restored contractility to 90% of baseline. Incontrast, in the control animal (dog B), pacing reduced contractility by40%, which did not improve with placebo infusion over the next 48 hours.Hence, GLP-1 markedly improves myocardial contractility afterpacing-induced heart failure (or hibernating myocardium).

[0058]FIG. 2 reflects changes in LV ejection fraction (EF), as measuredby percent emptying of the LV during systole. In the treated animal (dogA), pacing reduced LVEF by 40%, which then improved to 88% and 95% ofthe baseline value after 24 and 48 hours of GLP-1 treatment,respectively. In the control animal (dog B), pacing reduced LVEF byabout 30%, which subsequently improved only modestly over the next 48hours. Hence, GLP-1 treatment improves LVEF after pacing-induced heartfailure.

[0059]FIG. 3 illustrates LV contraction, as reflected by the degree ofwall thickening. In the treated animal (dog A), pacing resulted in a 20%reduction of wall thickening, which recovered after 24 hours of GLP-1treatment and actually increased to 147% of the baseline value after 48hours of treatment. In contrast, in the control animal (dog B), wallthickening was reduced by 25% after pacing, and this declined further to62% of the baseline value over the 48-hour placebo treatment period.Hence, GLP-1 treatment markedly improves LV contraction afterpacing-induced heart failure.

[0060]FIG. 4 reflects changes in overall cardiac function, as measuredby cardiac output (CO), which is the volume of blood (in mL) pumped perminute. CO is a product of stroke volume (volume of blood in mL expelledper systolic contraction) and heart rate (beats per minute). CO is areflection of myocardial contractility (i.e., the intrinsic force ofcontraction) as well as of systemic hemodynamics, including pre-load(i.e., venous filling pressures) and after-load (i.e., mean arterialpressure and systemic vascular resistance). In the treated animal (dogA), pacing resulted in a 30% reduction of CO, which was restored tobaseline levels after 24 hours of GLP-1 treatment, and actuallyincreased to 116% of baseline after 48 hours of treatment. In contrast,in the control animal (dog B), CO only fell by 7% after pacing, whichmay indicate that in this particular animal there was hemodynamiccompensation for the reduced myocardial contractility (FIG. 1) and LVEF(FIG. 2), thereby maintaining CO near normal. Nevertheless, over the48-hour placebo treatment, CO declined further, to 89% of baseline.Hence, GLP-1 treatment markedly improves cardiac output afterpacing-induced heart failure. TABLE 2 GLP-1 CONTROL BEFORE AFTER BEFOREAFTER NE 2.30 ± 0.15  1.62 ± 0.11* 1.55 ± 0.37  1.87 ± 0.14* (nmol/ml)LVP 98 ± 2  108 ± 2*  109 ± 4  104 ± 2  (mm Hg) LVEDP 25 ± 1  15 ± 1* 25± 2  21 ± 2  (mm Hg) dP/dt 1127 ± 86  2212 ± 86*  1650 ± 100  1736 ±112  (mm Hg/s) CO 1.38 ± 0.15  1.82 ± 0.12* 1.60 ± 0.10 1.42 ± 0.14(ml/min) CBF 27 ± 1  37 ± 3* 33 ± 3  33 ± 1  (ml/min) MVO_(2 (ml) 246 ±18  297 ± 16* 280 ± 38  287 ± 23  O₂/min

What is claimed is:
 1. A method for treating hibernating myocardium,comprising administering a therapeutically effective amount of a GLP-1molecule to said patient.
 2. A method for treating a patient sufferingfrom congestive heart failure, comprising administering atherapeutically effective amount of a GLP-1 molecule to said patient,wherein said patient also has hibernating myocardium.
 3. A method oftreating a patient suffering from ischemic cardiomyopathy, comprisingadministering a therapeutically effective amount of a GLP-1 molecule tosaid patient, wherein said patient also has hibernating myocardium.
 4. Amethod of treating a patient suffering from diabetic cardiomyopathy,comprising administering a therapeutically effective amount of a GLP-1molecule to said patient.
 5. The method according to any one of claims 1through 4, wherein the administration is continuous.
 6. The methodaccording to any one of claims 1 through 4, wherein the administrationis parenteral.
 7. The method according to any one of claims 1 through 4,wherein said effective amount of GLP-1 is effective to cause a reductionin the plasma or heart norepinepherine level.
 8. A method of decreasingthe plasma or heart norepinephrine level to a patient in need thereof,comprising administering to said patient a therapeutically effectiveamount of a GLP-1 molecule.
 9. A method of any one of claims 1-4 or 8,wherein said GLP-1 molecule is GLP-1 (7-36) amide.